Nanodrop 2000c spectrophotometer

Total RNA of the pericarp from self-rooted, grafted, and failed grafted cucumber (cucumber scion which developed roots connected with the stock pumpkin and/or raised new roots into the soil) with three biological replicates were extracted using a DNeasy Kit and miRNeasy Kit, respectively (QIAGEN, USA). The concentration and quality of DNA and RNA were evaluated by a NanoDrop 2000C Spectrophotometer and an Agilent 2100 Bioanalyzer. Pyro-sequencing assays were designed and performed by BIOMARKER Company with both programs and assay result data supplied. mRNA was isolated by Oligo-dT magnetic beads from RNA, then the cDNA was synthesized using a QiaQuick PCR Extraction Kit (QIAGEN, USA). The cDNA library was constructed and sequenced by Illumina Hiseq 2500.

Total RNA was extracted from the gonads (n = 6; 2 pools of 10 pairs of gonads per tank, 3 tanks per condition) using TRIzol reagent (Invitrogen, Life Technologies Europe B.V., Ghent, Belgium) as described previously (Baron 2005). The total RNA concentration was determined using an ISOGEN NanoDrop 2000c spectrophotometer (Wilmington, Delaware, USA) and RNA quality was controlled on a Bioanalyzer 2100 (Agilent). Only samples with a RIN (RNA Integrity Number) >8 were kept for further analysis. Labeled cDNA (using Cyanine-3) was synthesised, purified, quantified and prepared for hybridisation following the Agilent protocol [38 ].

Whole-genome sequences of S. cerevisiae strains Sc57 and in Sc57-Te₅^R were determined by Next Generation Sequencing (NGS). Preparation of indexed libraries and whole-genome shotgun sequencing were performed as a service by Genomix4Life S.r.l. using an Illumina platform. Samples were assessed after dilution by using Nanodrop 2000c spectrophotometer and Agilent 4200 TapeStation. After sequencing and quality check control of raw sequence data, paired-end reads were aligned against reference genome of strain S. cerevisiae S288C^{23 (link)} to identify single nucleotide variants (SNVs) and small insertions and deletions (InDels) using gold standard procedures. All SNVs abd InDels are listed in Supplementary Datasets S1 and S2. Alignments were performed with BWA^{55 (link)}, and SAMtools^{56 (link)} and BEDtools^{57 (link)} were used for filtering steps and file formats conversion. Reference genome of strain S. cerevisiae S288C (assembly R64) was used for functional annotation. Mapping of SNVs and InDels on S. cerevisiae coding sequences (CDSs) and intergenic genome regions, and classification of variants based on their effects on CDSs were obtained by using ad hoc developed Python programs.

Collected biopsy samples were immediately frozen and kept on −80 °C until use. Total RNA was isolated with the Qiagen miRNeasy Micro Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions, quantified on a NanoDrop 2000c Spectrophotometer and RNA integrity numbers (RIN) were calculated for all samples on an Agilent BioAnalyzer 2100 instrument.

The pink-flowered strawberry cultivar “Sijihong” and released by Shenyang Agricultural University in China was used in this study. According to the degree of flower opening and the change in petal color, the developmental stages were categorized into the young bud stage (PF_L), the coloration beginning stage (PF_Z), and the big bud stage (PF_D; Figure 1A; Xue et al., 2019 (link)). Petals of these three developmental stages with obviously different colors were collected for miRNA and degradome analyses, with three biological replicates per stage. The pink-flowered strawberry cultivar “Fenyun” was used to perform transient expression of candidate miRNAs and determination of anthocyanin contents. All samples were immediately frozen in liquid nitrogen and stored before −80°C for RNA extraction.
Total RNA was extracted using a modified CTAB method and treated with RNase-free DNase I (Takara, Dalian, China) to remove genomic DNA contamination. The RNA purity was measured by agarose gel (2.0%) electrophoresis. The Nanodrop 2000c spectrophotometer and Agilent 2100 were used to quantify the RNA amount and assess the RNA integrity with RIN number > 7.0, respectively.

At 30 DAP, seeds from individual plants in the BC₉ population were collected and stored in liquid nitrogen. The seed color of each individual plant was recorded when the seeds were mature, and the seeds were categorized into yellow-seed (BY) and brown-seed (BY) bulks. Prior to RNA extraction, the seeds were pooled, with each pool consisting of 30 seeds from lines of BY and BB bulks, respectively. The number of BC₉ lines in each bulk is listed in Table S1, and each bulk comprises 3 biological replicates for RNA sequencing (BSR-Seq). An RNA prep Pure Plant Kit (TIAN GEN, Beijing, China) was used for RNA extraction from each sample following the manufacturer’s instructions. The RNA concentration and quality were checked using a NanoDrop 2000c Spectrophotometer and an Agilent Bioanalyzer (RIN) for each sample. The mRNA was isolated and concentrated using magnetic beads attached with oligo d(T) for cDNA library preparation. The first-strand cDNA was synthesized from the mRNA using random hexamers. The cDNA libraries were prepared by ligating the cDNA fragments to the Illumina adapter followed by PCR amplification and purification with AMPure XP beads. The libraries were sequenced on an Illumina HiSeq 2000 platform with paired-end 150 bp reads by the SAGENE Company in Guangzhou, China.